Impacts of humic substances, elevated temperature, and UVB radiation on bacterial communities of the marine sponge Chondrilla sp

Abstract Sponges are abundant components of coral reefs known for their filtration capabilities and intricate interactions with microbes. They play a crucial role in maintaining the ecological balance of coral reefs. Humic substances (HS) affect bacterial communities across terrestrial, freshwater, and marine ecosystems. However, the specific effects of HS on sponge-associated microbial symbionts have largely been neglected. Here, we used a randomized-controlled microcosm setup to investigate the independent and interactive effects of HS, elevated temperature, and UVB radiation on bacterial communities associated with the sponge Chondrilla sp. Our results indicated the presence of a core bacterial community consisting of relatively abundant members, apparently resilient to the tested environmental perturbations, alongside a variable bacterial community. Elevated temperature positively affected the relative abundances of ASVs related to Planctomycetales and members of the families Pseudohongiellaceae and Hyphomonadaceae. HS increased the relative abundances of several ASVs potentially involved in recalcitrant organic matter degradation (e.g., the BD2-11 terrestrial group, Saccharimonadales, and SAR202 clade). There was no significant independent effect of UVB and there were no significant interactive effects of HS, heat, and UVB on bacterial diversity and composition. The significant, independent impact of HS on the composition of sponge bacterial communities suggests that alterations to HS inputs may have cascading effects on adjacent marine ecosystems.


Introduction
Sponges are abundant and speciose components of cor al r eef ecosystems, w ell kno wn for their filtr ation ca pacity (Diaz andRützler 2001 , De Goeij et al. 2013 ).They host diverse associations of micr obial comm unities , including viruses , arc haea, fungi, pr otozoa, and bacteria (Taylor et al. 2007, He et al. 2014, Pascelli et al. 2018 ).Most marine bacteria remain uncultured with inferred roles based on genomics or compound-uptake experiments .T hese studies support the potential importance of bacterial symbionts for nutrient acquisition and defense mechanisms (Selvin et al. 2010, Moitinho-Silva et al. 2017a, Burgsdorf et al. 2022 ).In the high microbial abundance (HMA) sponge Aplysina aerophoba , e.g.microbes accounted for the majority (65%-87%) of its dissolved organic carbon assimilation (Rix et al. 2020 ).Autotrophic photosymbionts , e .g. Ca.Synechococcus spongiarum , of the Indo-Pacific sponge Theonella swinhoei (Burgsdorf et al. 2022 ), in turn, w ere sho wn to transfer their photosynthates to inner sponge layers (Burgsdorf et al. 2022 ).The bacterial contribution to host defense is inferred fr om their pr oduction of a wide v ariety of bioactiv e secondary metabolites (Mohan et al. 2016 ).
Se v er al sponge species have shown relatively little change in bacterial composition across space and time (Erwin et al. 2015, Campana et al . 2021 ), although some other studies have found that the bacterial communities of a number of sponge species appear to be structured by spatial and environmental parameters (Olson et al. 2014, Busch et al. 2020, Cleary et al. 2022 ).Short-term heat exposure experiments , e .g., significantly impacted the bacterial composition of two sponge species: the common blue aquarium sponge, Lendenfeldia chondrodes , and the red excavating sponge Rhopaloeides odorabile (Webster et al. 2008, Fan et al. 2013, Vargas et al. 2021 ).In R. odorabile , this bacterial comm unity alter ation was accompanied by host-tissue deterioration (Webster et al. 2008, Fan et al. 2013 ), while in L. chondrodes , no tissue damage or bleaching was observed (Vargas et al. 2021 ).Periods of elevated temperatur es ar e often accompanied by intense UVB radiation and have been linked to El Niño southern Oscillation e v ents (Hughes et al. 2017 ).As these e v ents ar e pr edicted to gr ow mor e fr equent and se v er e (Ying et al. 2022 ), the future state of cor al r eef ecosystems will be determined by how they respond to these environmental disturbances .T he inter activ e effects of ele v ated temper atur es and UVB on sponge bacterial communities remain, ho w ever, less studied.
In addition to climatic factors, marine ecosystems have also been affected by changes to the quality and quantity of terrestriall y deriv ed or ganic inputs (Felgate et al. 2021, Curr a-Sánc hez et al. 2022 ).Terr estriall y deriv ed dissolv ed or ganic matter (tDOM) mainly enters the marine environment via underwater cave systems or river runoff and is found in gr eater concentr ations near coastal ecosystems (Esham et al. 2000 ).Humic substances (HS) are a major component of tDOM; these are complex organic compounds mainly formed through the decomposition of plant organic matter in terrestrial ecosystems (MacCarthy 2001 ).In the water column, HS provide UV-protective properties to reef organisms by absorbing sunlight in the ultraviolet (UV) spectrum (280-320 nm) (Ferrier-P a gès et al. 2007, Ayoub et al. 2012, Sharpless et al. 2014 ).Mor eov er, HS hav e been shown to promote bacterial diversity and the growth of potentially beneficial bacteria in fish (Louvado et al. 2021 ), and fav or the gro wth of particular bacterial groups within marine bacterioplankton communities (Nardi et al. 2021 ).
The quantity and quality of tDOM entering marine ecosystems has shifted due to changes at the terrestrial-aquatic interface.Conversion of natural wetlands and coastal forests to urbanized ar eas and a gricultur al fields has led to incr eased inputs of nutrients and pollutants, at the expense of natural inputs of natural organic substances including HS (Spaccini et al. 2006, dos Santos et al. 2019 ).Soils of deforested agricultural fields , e .g., produce 38%-53% less HS than natural forests soils (dos Santos et al. 2019 ).Although pr e vious studies hav e highlighted the importance of DOM in structuring sponge microbial communities (De Goeij et al. 2013, Campana et al. 2021, Shore et al. 2021 ), the specific effects related to the reduction and modification of HS in adjacent marine ecosystems, and sponges in particular, are largely unknown.
In this study, we used an experimental life support system (ELSS) to investigate the independent and interactive effects of HS supplementation, ele v ated temper atur e, and UVB r adiation on the bacterial communities associated with the tropical sponge Chondrilla sp.(Chondrillida: Chondrillidae).The r esults pr esented her e are an in-depth analysis of an earlier study, which discussed multiple cor al r eef biotopes (Stuij et al. 2023b ).Species of the genus Chondrilla are abundant on reefs in both tropical and temperate oceans (Usher et al. 2004 ).Chondrilla spp.have been shown to associate with a diverse array of microbial symbionts, including Cyanobacteria, Bacteriodetes, Acidobacteria, and Proteobacteria (Usher et al. 2001, Hill et al. 2006, Thiel et al. 2007 ).High microbial abundance (HMA) status has been suggested for Chondrilla spp.from the Caribbean and the Indo-Pacific, including C. australiensis , C. caribensis , and C. nucula (Usher et al. 2001, Hill et al. 2006, Alexander et al. 2014 ).W ith Chondrilla sp .as a model organism, we tested the hypothesis that the individual and inter activ e effects of HS, ele v ated temper atur e, and UVB will affect the composition and diversity of sponge bacterial communities.

ELSS design
The structure of the ELSS developed in this study was based on a microcosm system previously developed to assess the effects of global climate change and environmental contamination on sediment communities (Coelho et al. 2013 ).This system was modified and validated to allow the evaluation of the effects of HS, UVB r adiation, and temper atur e on host or ganisms and their associ-ated micr obial comm unities under labor atory-contr olled conditions (Stuij et al. 2023a ,b ).The ELSS included two frames of 16 glass aquaria eac h (r eferr ed to as microcosms, 23 cm in height, 16 cm length, and 12 cm width), which were individually connected to other aquaria (r eferr ed to as reservoirs, 30 cm in height, 12 cm length, and 12 cm width).Eac h r eservoir-micr ocosm unit contained a functional w ater v olume of ∼5 l.The microcosms and reservoirs contained outflow-holes (two cm in diameter) positioned 3 cm and 5 cm below the top of the glass, r espectiv el y ( Figure S1 , Supporting Information ).Water was circulated within micr ocosm-r eservoir units using small hydraulic pumps, resulting in a constant flow rate of ∼8.64 ± 0.66 ml s −1 .Water temper atur e was regulated using water bath tanks, each surrounding four microcosms, equipped with aquarium heaters with an internal thermostat (V2Therm 100 Digital heater, 100 W).Constant aeration within microcosms was maintained using diffuser stones (1.5 cm in diameter, 3 cm in length) connected to an air pump (530 L/h, Resun) via small hoses.Lighting was controlled by four fully programmable luminaire systems (Reef-SET, Rees, German y), eac h holding eight fluorescent lamps (Coelho et al. 2013 ).During the experiment, four UV fluorescent tubes (Solar-Raptor, T5/54 W, Rees, Germany) and four full spectra fluorescent tubes (ATI AquaBlue Special, T5/54 W) were connected alternately and programmed to a 12-h diurnal light cycle, simulating photoperiod conditions of tropical latitudes.To block radiation in the UVB spectrum (290-320 nm), micr ocosms wer e cover ed r andoml y by tr anspar ent pol yester films (Folanorm SF-AS, Folex coating, Köln, Germany).The film has been used in multiple studies, which evaluated UVB radiation (Müller et al. 2009, Rautenberger et al. 2013 ).In our experimental set-up, the film absorbed 90% of the UVB, 31% of UVA, and 9% of PAR irradiance.Detailed information on the light energy transmitted during the day can be found in Supplementary material 1 and Figure S2 ( Supporting Information ).
A multifactorial experiment was designed to test for the inde pendent and interacti ve effects of HS supplementation, temper atur e and UVB r adiation.Eac h factor had two le v els, namel y, HS supplementation (with versus without), temperature [normal ( ∼28 • C) versus heat ( ∼32 • C)] and UVB radiation (with versus without) for a total of eight conditions with four replicates each.T he full experiment, thus , consisted of 32 microcosms .T he temper atur e tr eatment was r andomized in gr oups of four micr ocosms; HS supplementation, UVB radiation and the combination of both were randomly assigned within each group of microcosms with equal temper atur e .T he eight conditions are abbreviated as follows: (1) Control , (2) UVB , (3) Heat , (4) UVB + Heat , (5) HS , (6) UVB + HS , (7) HS + Heat , and (8) HS + UVB + Heat .
Eac h micr ocosm was spiked with a coral reef sediment layer of ∼3 cm, consisting of a mixture of commercially available (Reef Pink dry ar a gonite sand, Red Sea) and natur al cor al r eef sediment.The commercial sediment was washed and sterilized three times (autoclavation at 121ºC for 20 min; Otte et al. 2018 ) before use.Natural sediment was collected from a coral reef environment at a depth of 6 m in the Penghu Islands (Taiwan), south of Fongguei (22 • 19 50.5 N 120 • 22 19.8 E), and used unprocessed.Synthetic seawater was added to the systems, pr epar ed by mixing cor al r eef salt (CORAL PRO SALT, Red Sea) with r e v erse osmosis gr ade water (V2Pur e 360) to a concentration of 35 ppt.HS were added to half of the microcosms as follows: a concentrated HS stock solution (10 g l −1 ) was first prepared by dissolving commerciall y av ailable HS (tec hnical gr ade humic acid, Sigma-Aldric h) using NaOH in deionized water.Subsequently, the solution was neutralized to a pH of 8.0-8.2 by adding concentrated HCL.Total carbon (TC), inorganic carbon (IC), organic carbon (TOC: TC-IC), and nitrogen (N) in the concentrated HS stock solution were measured using IR detection on a Multi N/C TOC anal yser (Anal ytica Jena; Table S1 , Supporting Information ).The TOC concentration in the stock solution equaled 45.82 ± 5.12 mg l −1 .This concentr ated stoc k solution was then used to enrich the synthetic seawater to a final HS concentration of 7.5 mg l −1 .Deriv ed fr om the stock solution measurements, this resulted in an addition of 2.83 ± 0.32 μmol T OC l −1 .T he concentration of TOC used in this experiment was in the range of terrestrial dissolved organic carbon (tDOC) pr e viousl y detected by Zhou et al. ( 2021 ) and Kaushal et al. ( 2022 ) in multiyear biogeochemical time series analyses of shallow cor al r eef waters in the central Sunda Shelf (Singa por e Str ait).
A total of 20% of the water in the r eservoir-micr ocosm unit was r ene wed eac h day by adding 1 l of fr eshl y pr epar ed synthetic seawater either with or without HS, to the r espectiv e microcosms.Stability of the HS concentration in the water column of the microcosms was monitored using UV spectrometry (Eaton 1995 ).The absorbance at a wavelength of 300 nm was relatively stable over the course of the experiment and av er a ged 0.017 ± 0.003 AU among microcosms .T he water was heated to 28 • C in all microcosms and the UVB absorbing films were applied to all microcosms.
After 8 days of initial system stabilization, we added one specimen of the sponge Chondrilla sp. to each microcosm.Alongside the sponge, we added one specimen of four other cor al r eef species into eac h micr ocosm.These included tw o har d corals, Montipora digitata (Dana, 1846) and Montipora capricornis (Veron 1985), one soft coral, Sarcophyton glaucum (Quoy & Gaimard, 1833) and one zoanthid, Zoanthus sp.The effects of HS, temper atur e, and UVB on these organisms and their microbial symbionts are discussed else wher e (Stuij et al. 2023b ).After introducing the organisms, the micr ocosms wer e maintained under the specified conditions for 21 days in order to acclimate the organisms to the microcosm conditions.All animals used in this study were obtained from the collection of marine inv ertebr ates cultiv ated at ECOMARE (University of Aveiro, Portugal).ECOMARE holds validated coral reef cultur e systems (Roc ha et al. 2015 ), whic h hav e pr e viousl y been used to study environmental effects on coral reef species under experimentall y contr olled conditions (Roc ha et al. 2020 ).These animals were cultivated at 26 • C, a salinity of ∼ 35 ppt, and pH of ∼ 8.2 (Rocha et al. 2015 ).
The Chondrilla sp.analyzed in the present study grew on the live r oc ks pr esent in the culture system collected fr om Indo-P acific reefs.It is an encrusting sponge with a br own-oliv e gr een color and cartilaginous consistency (Fig. 1 ).Identification of the specimen to the genus Chondrilla (family Chondrillidae) was done by N .J. de V oogd.Species of the genus Chondrilla possess a r elativ el y simple skeleton and few spicule types (Usher et al. 2004 ).So far, 16 valid Chondrilla species are known worldwide and four have been described from Indonesia, these are Chondrilla australiensis Carter, 1873, Chondrilla grandistellata T hiele , 1900, Chondrilla jinensis Hentschel, 1912, and Chondrilla mixta Schulze, 1877(Putra et al. 2023 ), but neither of them fitted the c har acters of our species pr ecisel y.The outer mor phology and skeletal featur es between species of Chondrilla ar e v ery similar and there are only a few characters that can be used to distinguish between species (Fromont et al. 2008 ).Giv en the abov e, we could not identify our specimen to species le v el and refer to the sponge studied here as Chondrilla sp.
We collected forty 1 cm 2 pieces fr om lar ger mother colonies, each with a single osculum.We used a scalpel to r emov e these pieces and then secur el y attac hed them to carbonate stones us-F igure 1. F r a gment of Chondrilla sp.used in the present study.
ing strong adhesive (100% c y anoacrylate).Follo wing this, these fr a gments wer e giv en 4 months to heal and firml y adher e to the ne w surface befor e being mov ed to the micr ocosms for further study.Befor e fr a gmentation, we made sur e the original sponge colony was healthy by observing its pumping activity with fluorescent dye.We verified the health of the sponge fr a gments by noting growth and attachment to the carbonate stones.A total of 32 cultiv ated sponges wer e ha phazardl y selected fr om the ECOMARE culture system and transplanted into the microcosms.Among the r emaining cultiv ated sponges at ECOMARE, four were utilized to analyze the sponge-associated bacterial communities under original culturing conditions (non-transplanted sponges).
After the acclimatization period, heat and UVB treatments wer e a pplied for 5 da ys as follows .In the heat-tr eated micr ocosms, temper atur e was gr aduall y incr eased ov er the course of 3 days from 28.0 ± 0.6 • C at day 1 to 31.0 ± 0.6 • C at day 3.In the UVBtr eated micr ocosms, the UVB absorbing film was r emov ed, whic h resulted in daily doses of 2.43 × 10 −2 J cm −2 UVB radiation during 5 da ys .A gr a phical summary of the different phases of the experiment can be found in Fig. 2 .

Physical and chemical parameters
Water temper atur e, pH, dissolv ed oxygen, and salinity (Multi 3420 multimeter, WTW GmbH, Weilheim, Germany) were periodically monitored during the acclimatization period (one measurement per day) and experimental phase (two measurements per day).Water samples for determining dissolved inorganic nutrient concentr ations (nitr ate NO

Sampling and DNA extraction
At the end of the experiment, all Chondrilla sp.specimens (32 sponges) were sampled from the microcosms.Upon collection, the sponges were photographed.These photos were used to estimate their size based on surface area in the software Image J .Four specimens were sampled from the ECOMARE facility to analyze the bacterial community composition of the sponges under their original culture conditions.All collected samples were washed with filtered and sterilized artificial seawater (with a pore size of 0.22 μm) and car efull y cut off their carbonate stones using a scalpel and subsequently weighed.The wet weight of the specimens and size upon collection varied between 0.06-0.60g and 1.92-5.99cm 2 .Controls for ELLS contamination with environmental DNA and sample collection were performed as previously described (Stuij et al. 2023a ).All samples were frozen at −80 • C until DNA extraction.An overview of the metadata, size and weight of the samples can be found in Table S2 ( Supporting Information ).PCR-ready genomic DN A w as isolated using the FastDNA ® SPIN soil Kit (MPbiomedicals) following the manufacturer's instructions.Due to the small size of Chondrilla sp., whole organisms were used for extraction.Blank negative controls, in which no tissue was added to the Lysing Matrix E tubes, were also included.Micr obial cell l ysis was performed in the FastPrep Instrument (MP biomedicals) for 2 × 40 s at a speed setting of 6.0 ms −1 .Extracted DN A w as eluted in 50 μl of DNase/pyr ogen-fr ee water and stored at −20 • C until further use.

16S rRNA gene library preparation and sequencing
The V3-V4 variable region of the 16S rRNA gene was amplified using primers 341F 5 CCTA CGGGNGGCWGCA G 3 and 785R 5 GA CTA CHV GGGTATCTAATCC 3 (Klindworth et al. 2013 ) with Illumina Nextera XT overhang adapters for a dual-PCR library pr epar ation a ppr oac h.PCRs wer e performed using 1-3 μl of DNA template, 10 μl of HS ReadyMix (KAPA HiFi Roche), and 0.6 μl of the forw ar d and r e v erse primers in a concentr ation of 10 pMol μl −1 .Reaction mixes were finalized by the addition of DNase free distilled water (Ultr a pur e , T hermoscientific) to a final volume of 20 μl.The PCR conditions consisted of initial denaturing at 95 • C for 3 min, follo w ed b y 30 c ycles of 98 • C for 20 s, 57 • C for 30 s, and 72 • C for 30 s, after which a final elongation step at 72 • C for 10 min was performed.We c hec ked for the success of amplification, r elativ e intensity of the bands, and contamination using 2% Invitrogen Egels with 3 μl of PCR product.
PCR pr oducts wer e cleaned with magnetic beads at a ratio of 0.9:1 using a ma gnetic extr actor stamp, after whic h a second PCR was performed.The 25 μl reaction mix consisted of 4 μl of the first PCR product, 12.5 μl HS ReadyMix (KAPA HiFi Roche), 2 × 1 μl (concentration of 10 pMol μl −1 ) MiSeq Nextera XT adapters (dual indexed, Illumina), and 6.5 μl of mQ water (Ultr a pur e).PCR conditions consisted of initial denaturing at 95 • C for 3 min, follo w ed b y eight c ycles of 98 • C for 20 s, 55 • C for 30 s, and 72 • C for 30 s, after which a final elongation step at 72 • C for 5 min was performed.DNA molarity and fr a gment sizes of the resulted PCR pr oducts wer e measur ed on a fr a gment anal yzer 5300 (Agilent) and subsequently normalized and pooled together using the Qiagen QIAgility.The pool of normalized DN A w as cleaned one last time using magnetic beads at a ratio of 0.65:1 and thereafter se-quenced at a commercial company (Baseclear, Leiden, the Netherlands) on an Illumina MiSeq platform using 2 × 300 bp pairedend sequencing (Illumina MiSeq PE300).Thr ee negativ e contr ol samples were included to detect possible contamination during libr ary pr epar ation and sequencing.Sequences fr om eac h end wer e paired following Q25 quality trimming and removal of short reads ( < 150 bp).The DNA sequences generated in this study can be downloaded from NCBI BioProject Id: PRJNA904682.Sample metadata, Biosample IDs and SRA numbers of the samples used in the current study are listed in Table S2 ( Supporting Information ).

Sequencing analysis
Dem ultiplexed gzipped FASTQ files, whic h contained pair ed forw ar d and r e v erse r eads for each sample, were imported and visualized using QIIME2 (Bolyen et al. 2019 ).Subsequently, forw ar d and r e v ersed sequences wer e truncated to a length of 245 nt and 200 nt, r espectiv el y, using the D AD A2 plugin (Callahan et al. 2016 ).The D AD A2 anal ysis pr oduced a quality filter ed table of all amplicon sequence variants (ASVs), a fasta file of re presentati ve sequences, and a table summarizing the denoising statistics.Following this, the QIIME2 feature-classifier plugin with the extractreads option was used to extract reads from the Silva database with the silva-138-99-seqs.qzafile as input and the forw ar d and r e v erse PCR primers as parameters .T his produced a file of reference sequence r eads, whic h was used as input for the featureclassifier plugin with the fit-classifier-naive-bayes option.ASVs generated by the D AD A2 analysis were classified taxonomically using the feature-classifier plugin in QIIME2 with the classifysklearn method.Mitoc hondria, c hlor oplasts, and Eukaryota were filtered out from the obtained ASV table using the QIIME2 taxa plugin with the filter-table method.The ASV count table is presented in Table S3 ( Supporting Information ).
Subsequentl y, we r emov ed arc haeal sequences, ASVs that were unassigned at the Domain and Phylum le v els, in addition to ASVs, whic h occurr ed in the triple-autoclav ed commercial sediment (control for sampling and eDNA contamination), and negativ e contr ols used for sequencing.ASVs r emov ed following detection in the control samples are listed in Table S4 ( Supporting Information ).Ov er all, the r emov ed ASVs were assigned to known contaminants , e .g., the gener a Ralstonia , Bur kholderia-Caballeronia-Par aburkholderia , Reyr anella , Bacillus , and Br adyrhizobium (Salter et al. 2014, Glassing et al. 2016, Weyrich et al. 2019 ).Abundant ASVs wer e r efer enced a gainst the NCBI nucleotide database using the NCBI Basic Local Alignment Search Tool (BLAST) (Zhang et al . 2000 ).BLAST identifies locally similar regions between sequences, compares sequences to extant databases and assesses the significance of matches.

Sta tistical anal ysis
A table containing ASV counts was imported into R and used to anal yse how tr ansplantation and the tr eatments influenced bacterial composition and higher taxon abundance.Differences in r ar efied ric hness (sample size of 15 556 sequences), e v enness (calculated by dividing Shannon's H' by the number of ASVs in each sample), and higher taxon abundances between ECOMARE and Control samples, and among treatments were investigated using an analysis of deviance using the glm() function of the R package stats.A number of these variables included an excess of zero counts in the samples, ther efor e, we set the family argument to "tweedie" using the tweedie function in R with var.power = 1.5 and link.pow er = 0 (a compound Poisson-gamma distribution).Using the glm model, we tested for significant variation using the anova() function in R with the F test.To correct for multiple testing in the r elativ e abundance of phyla, classes, and or ders, w e applied the Bonferr oni corr ection.This corr ection accounts for type I err ors and compar es P-v alues to α n , in whic h α r epr esents the threshold significance level and n the number of tests.For each taxonomic le v el, we anal yzed if ther e was an effect in the four most abundant gr oups, r esulting in four tests per taxonomic le v el and a corrected significance le v el of 0 . 054 = 0 .0125 .Variation in bacterial composition was visualized with principal coordinates analysis (PCoA).For the PCoA, the ASV table was r ar efied to the minimum sample size (15 556 sequences) using the rr ar efy() function of the R pac ka ge v egan.For compositional anal yses, the ASV table was log( x + 1) transformed (in order to normalize the distribution of data) and a distance matrix constructed using the Bray-Curtis index with the vegdist() function in the vegan package in R. Subsequently, we used the cmdscale() function of the R pac ka ge stats with the Bray-Curtis transformed distance matrix as input.We tested for significant differences in ASV composition with a perm utational anal ysis of v ariance (PERMANOVA) using the ado-nis2() function and for homogeneity of m ultiv ariate dispersion using the betadisper() function of the R pac ka ge v egan (999 perm utations).Detailed descriptions of the functions used here can be found in R and online in r efer ence manuals of pac ka ges (e.g.http: //cr an.r pr oject.org/web/pac ka ges/v egan/index.html ).For the factors that significantly explained variation in our dataset, we calculated the effect size ( ω2) using the function adonis_OmegaSq(), with the adonis2 test result as input.The source code of this function is given in the Supplementary material .The function is based on the MicEco: adonis_OmegaSq() function of the pac ka ge MicEco but adjusted so it works with adonis2.
To identify specific classes, orders and ASVs that associated with giv en tr eatments, we used a featur e selection algorithm called "Boruta."Boruta, named after a slavic forest demon, is a r andom for est wr a pper, whic h is used to e v aluate featur e importance (Kursa et al. 2010 ).Boruta iter ativ el y compar es the importance of features with the importance of shadow features, created by shuffling the original ones .F eatures that ha v e significantl y less importance than shadow ones are consecutively dropped.On the other hand, features that are significantly better than shadows are identified as confirmed.It is assumed that confirmed features with the highest importance are most relevant to the outcome variable.In the present study, the Boruta() function from the Boruta pac ka ge in R was used with HS and Heat as r esponse v ariables and the r elativ e abundance of bacterial classes , orders , and ASVs as features (predictive variables).The doTrace argument in the Boruta() function was set to 2 and the maxRuns argument set to 1000; other arguments used default v alues.Onl y ASVs with an absolute abundance of > 100 sequences across the dataset were included (58 ASVs).Significant predictor ASVs wer e r efer enced against the NCBI nucleotide database using NCBI BLAST (Zhang et al. 2000 ).

Physical and chemical parameters
The results of the water quality analysis are summarized in Figures S3 -S6 ( Supporting Information ).During the acclimatization phase, temper atur es r anged fr om 26.4 ± 0.6 • C in the morning to 27.6 ± 0.8 • C in the afternoon.Salinity av er a ged 35.50 ± 0.74 ppt.pH varied from 8.10 ± 0.05 in the morning to 8.21 ± 0.07 in the afternoon for HS , and from 8.05 ± 0.06 to 8.16 ± 0.07 for Control micr ocosms.Dissolv ed oxygen le v els r anged from 8.12 ± 0.21 mg l −1 in the morning to 8.39 ± 0.34 mg l −1 in the afternoon for HS , and from 8.15 ± 0.34 mg l −1 to 8.55 ± 0.44 mg l −1 for Control microcosms.During the experimental phase (see Figure S4 , Supporting Information ), microcosms without heat treatment had temperatures of 27.2 ± 0.5 • C in the morning and 28.0 ± 0.6 • C in the afternoon.In heat-treated microcosms, temper atur e incr eased gr aduall y ov er 3 days from 28.0 ± 0.6 • C on day 1 to 31.0 ± 0.6 • C on day 3, av er a ging 31.4 ± 0.5 • C and 31.2 ± 0.7 • C on days 4 and 5, r espectiv el y.Dissolv ed oxygen le v els r anged fr om 7.01 ± 0.40 mg l −1 in Heat + UVB + HS in the morning to 9.06 ± 0.36 mg l −1 in UVB microcosms in the afternoon.pH ranged from 8.09 ± 0.05 in Heat microcosms in the morning to 8.32 ± 0.024 in UVB microcosms in the afternoon.
Por e water TOC concentr ations pr ogr essiv el y r ose thr oughout the experiment.No independent or inter activ e effect of our factors explained significant variation in the dataset (PERMANOVA using adonis2 in the R pac ka ge v egan, P > .05).The mean values were 0.14 ± 0.01 mmol C l −1 on day 8, 0.25 ± 0.1 mmol C l −1 on day 28, and 0.41 ± 0.24 mmol C l −1 on day 34.

The effect of transplantation on sponge bacterial communities
The bacterial composition and diversity of specimens obtained from the ECOMARE facility and their variance from the control specimens collected from the microcosms, are depicted in Figures S7 and S8 ( Supporting Information ).Bacterial richness and e v enness did not differ significantly between ECOMARE and microcosm sponges (GLM, P > .05,Table S6 , Supporting Information ).Bacterial community composition, ho w ever, significantly differed between ECOMARE and microcosm sponges (PERMANOVA: F 1,6 = 5.72, R 2 = 0.489, P = .021,ω 2 = 0.43, PERMDISP: F 1,6 = 1.26,P = .21).This variation was visualized in an ordination analysis ( Figure S7b , Supporting Information ) based on bacterial composition, whic h separ ated samples collected from ECOMARE and the microcosms on the first axis of v ariation.We subsequentl y inv estigated if variation occurred in higher taxonomic composition (four most abundant phyla, classes, and orders), and found some minor, non significant variance in the relative abundances of the four most abundant phyla and classes (see Figure S7c , Supporting Information ).Of the four most abundant or ders, HOC36 w as significantl y mor e abundant in ECOMARE samples than in the microcosm controls (GLM, P = .012;Table S6 , Supporting Information ).Sponges of both ECOMARE and the microcosms were dominated by the same group of ASVs ( Figure S8 , Supporting Information ).

Sponge bacterial communities under the independent and interacti v e effect of HS, heat, and UVB
After quality control and r emov al of ASVs assigned to c hlor oplasts and mitochondria, and ASVs unassigned at Domain and Phylum le v el, the dataset consisted of 875 721 sequences and 397 ASVs.A full ov ervie w of the observ ed phyla, classes and orders and their abundances is presented in Table S5 ( Supporting Information ).In terms of sequences, the most abundant phyla were Proteobacteria, Cyanobacteria, Acidobacteriota, Gemmatimonadota, Chloroflexi, and Actinobacteriota (all > 20 000 sequence reads).The phyla with the highest numbers of ASVs wer e Pr oteobacteria, Verrucomicrobiota, Bacteriodota, Planctomycetota, and Cyanobacteria (all > 20 ASVs).

Bacterial di v ersity and composition
Ther e wer e no significant differ ences (GLM, P > .0125)among treatments in any of the diversity indices.Bacterial richness varied from 58.96 ± 4.73 in Heat + UVB to 85.52 ± 19.43 in Heat .Evenness varied from 0.61 ± 0.03 in Heat to 0.68 ± 0.05 in UVB microcosms (Fig. 3 ; Table S6 , Supporting Information ).

Significant predictor higher taxa
The Boruta analysis detected six classes and 11 orders as significant predictors of HS, whereas one class and tw o or ders w ere found to be significant predictors of Heat ( Table S7 , Supporting Information ).The r elativ e abundances of the orders with the highest importance values (importance value > 5.00%) are presented in Fig. 6 .Of these, the orders BD2-11 terr estrial gr oup and Sacc harimonadales wer e r elativ el y mor e abundant and, the orders Nitrosococcales , Dadabacteriales , Steroidobacterales , Puniceispirillales , and T halassobaculales r elativ el y less abundant in HS-supplemented microcosms .T he or der Planctomycetales w as the only significant and positive predictor of Heat with an importance value > 5.00% ( Table S7 , Supporting Information ).

Significant predictor ASVs
Among the 30 most abundant ASVs, Boruta analysis detected 10 significant predictors of HS and one significant predictor of both HS and Heat (Fig. 7 , Tables S7 and S8 , Supporting Information ).Among the less abundant ASVs, we detected five significant predictors of HS, one of Heat and one of both HS and Heat.Taken together, the abundances of these ASVs accounted for > 96% of all sequences.

Discussion
In this study, we used an ELSS to investigate the independent and inter activ e effects of ele v ated temper atur e, UVB r adiation, and HS-supplementation on the bacterial communities of the tropical sponge Chondrilla sp.This model system allo w ed us to test hypotheses with respect to an impact of these factors on bacterial diversity and composition with a high level of experimental contr ol and r eplication, whic h would be impossible to ac hie v e with field studies alone.

Physical and chemical conditions in the microcosms
Av er a ge v alues of water temper atur e, dissolv ed oxygen, pH, and salinity in the microcosms were similar to conditions measured at shallow coral reef sites (Guadayol et al. 2014, Bainbridge 2017, De-Carlo et al. 2017 ).During both the acclimatization (see also Stuij et al. 2023a ) and experimental phase, we observ ed dail y fluctuations in oxygen and pH (higher in the morning and lo w er in the afternoon).This variation is common at reef sites and is a result of netphotosynthesis during the day and r espir ation at night (Guadayol et al. 2014 ).During the experimental phase, oxygen le v els wer e lo w er in heated micr ocosms, whic h can be attributed to the positiv e corr elation between or ganismal metabolic r ates and temperatur e (Br own et al. 2004 ).The effect of temper atur e w as ho w e v er marginal.The lo w est values w ere observed in Heat + UVB + HS microcosms but still averaged 0.22 ± 0.013 mmol O 2 l −1 .The highest v alues wer e observ ed in UVB micr ocosms in the afternoon and av er a ged 0.28 ± 0.01 mmol O 2 l −1 .At r eef sites, dissolv ed oxygen concentr ations typicall y r ange fr om 50% air satur ation to 200% air satur ation (corr esponding to 0.12-0.43mmol O 2 l −1 at 27 • C), depending on location and time of day (Nelson and Altieri 2019 ).
Por e water sulphate concentrations of surface sediments have been observed to vary between 23.96 and 30.00 mmol SO 4 2 − l −1 (Alongi 1995 , Werner et al. 2006 ).At the first time point, sulphate was r elativ el y high (30.75 ± 0.34 mmol l −1 ), but fell to le v els Relative abundances are grouped per treatment.Note that factor-related significant variation is indicated with an * (GLM, P < .0125).See Table S5 ( Supporting Information ) for detailed statistical results.).Note that the membrane pore size of our samplers was 0.6 μm, which included a slightly larger fraction of total organic matter compared to the measurements taken at the Great Barrier Reef.
The effect of transplantation on the bacterial communities of Chondrilla sp.
The transplantation of the sponges affected bacterial composition, indicating a certain le v el of micr obial ada ptation to the ne w en vironmental conditions .Sponges of both ECOMARE and the micr ocosms wer e dominated by the same group of ASVs ( Figure S8 , Supporting Information ; see also Stuij et al. 2023a ).
The independent and interacti v e effect of HS, heat, and UVB on the bacterial communities of Chondrilla sp.
Our r esults r e v ealed the pr esence of a cor e bacterial comm unity, composed of highly prevalent ASVs across all host individuals and whic h r emained r elativ el y abundant irr espectiv e of the envir onmental conditions.Ho w e v er, we also detected bacterial populations that a ppear ed mor e r esponsiv e to the independent effects of HS and temper atur e, explaining 14% and 5% of the observ ed v ariation in community composition, respectively.In line with the presence of a r elativ el y stable core community, Thiel et al. ( 2007 ) and Erwin et al. ( 2015 ) observed high host-specificity, but low seasonal and interannual compositional variation in bacterial communities of a number of HMA and LMA sponge species, including C. nucula , Dysidea avara , Chondrosia reniformis , Agelas oroides , Axinella damicornis , Petrosia ficiformis , and Spirastrella cunctatrix .
ASVs 1 and 4, assigned to Ca. S. spongiarum , and to Albidovulum sp., r espectiv el y, wer e stable cor e members.In congruence with our r esults, pr e vious studies sho w ed that members of the Ca. S. spongiarum group remained abundant in sponge hosts ( Iricinia variabilis and Xestospongia muta ) across a range of temperatures and different irradiance regimes (Erwin et al. 2012, Lesser et al. 2016 ).Albidovulum members have previously been found in marine and terrestrial hot springs (Albuquerque et al. 2002, Yin et al. 2012 ) suggesting that members of this genus are heat-tolerant.Other abundant ASVs common to all treatments were assigned to the Sv a0996 gr oup , PA UC26f group , and Rhodobacter aceae famil y, all of whic h hav e been fr equentl y found in association with HMA sponges (Moitinho-Silva et al. 2017b, Turon et al. 2018 ).
In contrast to the abo ve , HS increased the relative abundances of the BD2-11 terr estrial gr oup, order Sacc harimonadales and eight ASVs assigned to the BD2-11 terrestrial and HOC36 groups, SAR202 and KI89A clades, Rhodobacteraceae and Hyphomonadaceae families, and Pseudohongiella genus.All of these ASVs wer e closel y r elated to bacteria pr e viousl y detected in sponges (94%-100% sequence similarities, Table S8 , Supporting Information ).SAR202 members collected from the sponge A. aerophoba were suggested to degrade dissolved organic matter in seawater, aiding nutrient acquisition in their host sponges (Bayer et al. 2018 ).Sponge-associated Rhodobacteraceae displayed the genomic potential to reduce nitrogen-containing aromatic compounds and utilize sulphated pol ysacc harides, potentiall y contributing to benthic biogeochemical cycling (Karimi et al. 2019 ).Although the metabolic capabilities of BD2-11 terrestrial group members associated with sponges are not fully understood, members of this gr oup hav e pr e viousl y been found to decompose recalcitrant soil organic matter, such as HS, in soils (Pascault et al. 2013 ).Similarl y, Sacc harimonadales members have previously been found in soil, where they are thought to play a k e y role in phosphorus cycling by converting organic phosphorus to inorganic forms (Wang et al. 2022 ).It is possible that these bacterial gr oups wer e favor ed ov er other bacterial populations in the HS-supplemented microcosms due to their common ability to degrade complex organic molecules.
The r elativ e abundances of the HOC36 gr oup, orders Nitr osococcales , Dadabacteriales , Ster oidobacter ales , Puniceispirillales , and Thalassobaculales were negatively associated with HS.More specifically, we detected nine ASVs assigned to the HOC36 group, JTB255 marine benthic gr oup (Ster oidobacter ales order), OM75 clade (Thalassobaculales order), TK17 group, Dadabacteriales order, Simkaniaceae family (Chlamydiales order), and the genera AqS1 (Nitrosococcales order), Constrictibacter (Puniceispirillales order), and Pseudohongiella (Oceanospirillales order), which were negativ el y associated with HS.These bacterial groups are often detected in low nutrient envir onments (Ra ppé et al. 1997, Nelson et al. 2014, Graham and Tully 2021 ) with metabolic profiles specialized to obtain nutrients fr om inor ganic sources.Nitr osococcales , e .g., includes marine bacterial ecotypes involved in the recycling of inorganic nitrogen (Semedo et al. 2021 ).Moreover, the gene r epertoir e of AqS1 members detected in the sponge Amphimedon queenslandica , indicated that they were capable of sulfur oxidation, carbon mono xide o xidation, and inorganic phosphate assimilation (Gauthier et al. 2016 ).JTB255 members hav e fr equentl y been found in marine sediments and metagenome analysis has suggested that they are involved in sulfur oxidation and carbon fixation (Mußmann et al. 2017, Hoffmann et al. 2020 ).
Pr e vious studies hav e suggested that sponges play crucial r oles in nutrient cycling and energy transfer within coral reefs (De Goeij et al. 2013, Alexander et al. 2014, Rix et al. 2016 ).Given the effect of HS on sponge-associated bacteria observed in the present study, it will be interesting to further explore the potential role of sponges in the transfer of nutrients obtained from terrestrially derived organic matter, and HS specifically, to the marine food-web.
Ele v ated temper atur e (Heat) positiv el y influenced the r elativ e abundances of the order Planctomycetales and two proteobacterial ASVs (8 and 29), assigned to the families Pseudohongiellaceae and Hyphomonadaceae.Note that this effect in Planctomycetales was seen in Heat and Heat + HS , but less so in UVB + Heat and UVB + Heat + HS .Additionally, ASV-29 was only detected in HStr eated micr ocosms and the effect of Heat, ther efor e, onl y holds true for Heat + HS and UVB + Heat + HS .Members of the order Planctomycetales hav e pr e viousl y been found in association with both abiotic and biotic biotopes in terrestrial and aquatic environments (Wiegand et al. 2018 ).In aquatic en vironments , Planctomycetales members often associate with phototrophs (macroand microalgae, and c y anobacteria) and have been sho wn to have the genomic potential to degrade algal-derived sulphate polysacc harides (Glöc kner et al. 2003, Bengtsson et al. 2012, Wegner et al. 2013 ) In contrast to the abo ve , ASV-1739 (KI89A clade) was less abundant in Heat and Heat + UVB tr eated micr ocosms.Sc hellenber g et al. ( 2020 ) reported the specific loss of KI89A clade members associated with the sponge Haliclona cnidata following experimental exposure to detrimental conditions (light exclusion and addition of antibiotics).The r elativ e abundances of KI89A clade members wer e also negativ el y affected by incr easing temper atur e in the temperate sponge A. oroides (Castro-Fernández et al. 2023 ).In addition to their association with sponges, KI89A clade members are common oligotrophic marine bacteria that do not usually grow in nutrient-ric h envir onments (Cho and Giov annoni 2004 ).Inter estingl y, our r esults sho w ed that ASV-1739 w as present in all Heat + HS and Heat + UVB + HS micr ocosms, whic h indicates HS might, to some extent, have mitigated the adverse effect of ele v ated temper atur e on this specific ASV.

Conclusion
Her e, we observ ed that Chondrilla sp.hosted a cor e bacterial community unaffected by experimental differences in temper atur e, UVB radiation or HS-supplementation, and consisting of members related to organisms obtained from other HMA sponges.Ho w e v er, Chondrilla sp. also hosted a variable bacterial community, affected by HS and, to a lesser extent, ele v ated temper atur e. Ov er all, both HS and ele v ated temper atur e significantl y modified bacterial community composition and taxon abundance, whereas no inter activ e or UVB-r elated effects wer e observ ed.Ele v ated temper atur e positiv el y affected the r elativ e abundances of the order Planctomycetales and ASVs of the proteobacterial families Pseudohongiellaceae and Hyphomonadaceae, but negativ el y affected the abundance of a potential symbiont of the KI89A clade.HS, in turn, positiv el y affected the r elativ e abundances of se v er al ASVs

Figure 2 .
Figure 2. Gr a phical r epr esentation of the experimental set-up, pr e viousl y displayed in Stuij et al. ( 2023a ).

Figure 3 .
Figure 3. Boxplots of the r ar efied ric hness and e v enness under the independent and combined effects of UVB, heat, and HS supplementation.Diversity measur es ar e gr ouped per tr eatment.

Figure 4 .
Figure 4. Boxplots of the r elativ e abundance of the four most abundant phyla, classes, and orders under the independent and combined effects of UVB, heat, and HS supplementation.Relative abundances are grouped per treatment.Note that factor-related significant variation is indicated with an * (GLM, P < .0125).See TableS5( Supporting Information ) for detailed statistical results.

F igure 5 .
Or dinations sho wing the first tw o axes of the principal coordinates analysis (PCoA) of bacterial ASV composition.The PCoA was generated using the cmdscale() function in the R base package.Prior to the PCoA, the raw data were log(x + 1) transformed and used to produce a distance matrix based on the Bray-Curtis distance with the vegdist() function in the R-pac ka ge v egan.pr e viousl y observ ed byAlongi ( 1995 ) andWerner et al. ( 2006 ) in cor al r eef envir onments.Although we detected r elativ el y low TOC in the ELSS at the beginning of the experiment (0.14 ± 0.01 mmol C l −1 ), TOC concentr ations incr eased to 0.41 ± 0.24 mmol C l −1 at day 34.These v alues wer e in the r ange of DOC concentr ations ( ∼ 0.25 to 0.67 mmol C l −1 ) in surface sediment pore water at Great Barrier Reef sites (measured using 0.4 μm membr ane filters; Lour ey etal.2001

Figure 6 .
Figure 6.Boxplots of the r elativ e abundance of the orders with more than 5.00% importance in the Boruta analysis with HS or Heat as response v ariable.Significant pr edictors for HS: Nitr osococcales , Dadabacteriales , BD2-11 terrestrial group, Saccharimonadales, Steroidobacterales, Puniceispirillales , and T halassobaculales .Significant predictor for Heat: Planctom ycetales.Relati ve abundances are grouped per treatment.

Figure 7 .
Figure 7. Mean r elativ e abundances of the 30 most abundant and significant pr edictor ASVs (17 for HS; 3 for Heat).Relativ e abundances ar e gr ouped per treatment.Symbols are proportional to the relative abundance of the respective ASV and color-coded following their class-level taxonomic assignment.ASVs are labeled with their lo w est taxonomic classification.Hs-and Heat-significant predictor ASVs are labeled at the right side of the figure.

Table 1 .
Results of the PERMANOVA anal ysis.Coef: coefficient, Df: degr ees of freedom, SumsOfSqs: sum of squares, and MeanSqs: mean squares.Significant test results are depicted in bold.